Medicament based on plant material of genus rauvolfia, genus eclipta and genus phyllanthus for treatment of liver disease, liver injury and / or hepatotoxicity

Abstract

A multi-herbal formulation for the preparation of a pharmaceutical composition for the treatment of liver disease, liver injury or hepatotoxicity in a subject in need thereof, the multi-herbal formulation comprising plant material in the form of hedyotis diffusa, eclipta alba and phyllanthus bitter.

Description

BACKGROUND OF THE INVENTION

[0001] The present application relates to the use of plant materials selected from the group comprising Rauwolfia spp., Eclipta spp. and Phyllanthus spp. in the preparation of a medicament for the treatment of liver diseases, liver injury and / or hepatotoxicity. Polyherbal formulations utilize the concept of positive interaction between herbs to produce a synergistic effect. Without being bound by any theory, the phytochemical constituents of one plant are activated in the presence of the constituents of another plant.

[0002] EP0 890 360B describes a polyherbal formulation for the treatment of conditions associated with hepatitis E and hepatitis B virus infections. The formulation contains 25 - 250 mg of extract of each of the following plants: Rheum emodi Wall., Phyllantus amarus Linn., Eclipta alba Hassk., Andrographis paniculate Nees and Picrorhiza Kurroa Royle ex Benth.

[0003] US 6,136,316 provides another polyherbal composition useful for the treatment of hepatitis E virus infections including acute liver failure. The formulation contains extracts of Rheum emodi Wall., Phyllantus amarus Linn., Eclipta alba Hassk., Andrographis paniculate Nees and Picrorhiza Kurroa Royle ex Benth.

[0004] However, existing formulations and compositions are complex mixtures, and as the number of ingredients increases, the manufacturing cost increases, the risk of adverse side effects increases, and more importantly, the risk of interaction between herbs increases, and such interaction is not synergistic and may actually impair the overall activity of the formulation by counteracting the individual actions of the ingredients.

[0005] In addition, existing formulations fail to adopt a holistic approach to treatment and generally focus only on a single aspect of the infection or disease. Liver diseases constitute a spectrum of conditions including viral hepatitis, alcoholic liver disease, non - alcoholic fatty liver disease (NAFLD), cirrhosis and liver cancer. The effectiveness of current treatments varies depending on the specific disease and its stage, and there are challenges and gaps in addressing these complex health problems.

[0006] For advanced liver diseases, especially cirrhosis and liver cancer, a major challenge lies in the limited treatment options. Usually, liver transplantation is the only viable option at present, highlighting the urgent need for alternative interventions.

[0007] Another obstacle in liver disease treatment is related to the side effects and tolerability of existing drugs. Certain drugs may cause severe side effects or have poor tolerability in some patients, leading to non-compliance with treatment regimens and hindering overall efficacy. In addition, concerns about drug resistance continue to threaten the sustained treatment effect.

[0008] The emergence of non-alcoholic fatty liver disease (NAFLD) as a global health problem further highlights the gaps in current treatment methods. Although lifestyle changes such as diet and exercise are the main recommendations, targeted drug interventions are significantly lacking.

[0009] There is a need to provide new polyherbal formulations to seek relief from or overcome one or more of the above problems and to provide new ingredients with previously unknown activities in the formulations for the treatment of liver diseases, liver injury and / or hepatotoxicity. Summary of the Invention

[0010] In one aspect of the present invention, there is provided a polyherbal formulation (PHF) comprising or consisting of the following ingredients:

[0011] a) A plant or plant part of the Eclipta family, especially Eclipta alba, in an amount of at least 30%, preferably at least 35% or 40%; and

[0012] b) A plant or plant part of the Phyllanthus family, especially a plant or plant part of Phyllanthus amarus, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0013] c) A plant or plant part of the Rauwolfia family, especially a plant or plant part of Rauwolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0014] The PHF is preferably liver-specific (i.e., liver PHF) for treating liver diseases, liver injury or hepatotoxicity in a subject in need thereof.

[0015] In one embodiment, with respect to the plants and plant parts, the PHF consists only of plants or plant parts of the Eclipta family, Phyllanthus family and Rauwolfia family.

[0016] Preferably, the PHF contains not more than 70%, preferably not more than 60%, of plants or plant parts of the Ecliptaceae family; not more than 30% of plants or plant parts of the Phyllanthaceae family; and not more than 36% of plants or plant parts of the Apocynaceae family.

[0017] In a more preferred embodiment of this aspect of the present invention, a PHF is provided that contains 43% to 46% of plants or plant parts of the Eclipta genus, 23% to 26% of plants or plant parts of the Phyllanthaceae family, and 29% to 32% of plants or plant parts of the Apocynaceae family.

[0018] In the most preferred embodiment of this aspect of the present invention, the plants or plant parts are plants or plant parts of Eclipta prostrata, Phyllanthus amarus, and Rauwolfia serpentina.

[0019] Preferably, the PHF of the present invention has a synergistic effect.

[0020] The plant can be the whole plant; the plant part can be one or more of fruits, seeds, rhizomes, buds, leaves, husks, barks, flower petals and petal extracts, peels, seed coats, and stems.

[0021] In each of the above embodiments, the PHF further comprises one or more secondary metabolites selected from flavonoids, saponins, coumestans, phenols, alkaloids, tannins, and lignans.

[0022] In another aspect of the present invention, there is provided the use of a polyherbal formulation in the preparation of a medicament for treating liver diseases, liver injury, or hepatotoxicity in a subject in need thereof, wherein the medicament comprises or consists of the following components:

[0023] a) Plants or plant parts of the Ecliptaceae family, particularly plants or plant parts of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0024] b) Plants or plant parts of the Phyllanthaceae family, particularly plants or plant parts of Phyllanthus amarus, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0025] c) Plants or plant parts of the Apocynaceae family, particularly plants or plant parts of Rauwolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0026] In another aspect of the present invention, there is provided a method for treating liver diseases, liver injury, or hepatotoxicity in a subject in need thereof, comprising administering a polyherbal formulation comprising or consisting of the following components:

[0027] a) Plants or plant parts of the Ecliptaceae family, especially plants or plant parts of Eclipta prostrata, with a content of at least 30%, preferably at least 35% or 40%; and

[0028] b) Plants or plant parts of the Phyllanthaceae family, especially plants or plant parts of Phyllanthus amarus, with a content of at least 15%, preferably at least 20% and at most 40%; and

[0029] c) Plants or plant parts of the Apocynaceae family, especially plants or plant parts of Rauvolfia serpentina, with a content of at least 25%, preferably at least 27%.

[0030] In another aspect of the present invention, there is provided a multi-herbal preparation for treating liver diseases, liver injury or liver toxicity in a subject in need thereof, which comprises the following components or consists of the following components:

[0031] a) Plants or plant parts of the Ecliptaceae family, especially Eclipta alba, with a content of at least 30%, preferably at least 35% or 40%; and

[0032] b) Plants or plant parts of the Phyllanthaceae family, especially plants or plant parts of Phyllanthus amarus, with a content of at least 15%, preferably at least 20% and at most 40%; and

[0033] c) Plants or plant parts of the Apocynaceae family, especially plants or plant parts of Rauvolfia serpentina, with a content of at least 25%, preferably at least 27%.

[0034] In each aspect related to treating liver diseases, liver injury or liver toxicity in a subject in need thereof, the PHF preferably comprises 43% to 46% of plants or plant parts of the Ecliptaceae family, 23% to 26% of plants or plant parts of the Phyllanthaceae family, and 29% to 32% of plants or plant parts of the Apocynaceae family.

[0035] From the following description given by way of example and with reference to the accompanying drawings, other aspects of the present invention and other embodiments of the aspects described in the previous paragraphs will become apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shows the cytotoxicity of PHF, especially the inhibition percentage of PHF. Specifically, it supports the IC 50 assay of PHF against epithelial cells. The IC 50 of PHF against the epithelial cell line was determined to be 46.21 μg / mL.

[0037] Figure 2 Shows the cellulose fiber content in PHF.

[0038] DEFINITIONS

[0039] “Polyherbal formulation” (PHF) refers to a preparation that uses more than one herb as an ingredient to enhance the therapeutic effect of individual herbs and reduce the toxicity of individual herbs. The term “formulation” can be used interchangeably with “composition”, and the abbreviation PHF can be used interchangeably with polyherbal formulation. It is intended specifically for the treatment of liver diseases, liver injury or hepatotoxicity in subjects in need thereof, and may be referred to herein as liver-specific PHF or liver PHF.

[0040] As used herein in the context of PHF, “synergy” refers to the combined effect of plant components being greater than the sum of the effects seen when each plant component is administered alone.

[0041] “Plants and plant parts” refers to the whole plant, fragments of the whole plant (such as the stem of the plant) or parts of the plant, including but not limited to fruits, seeds, rhizomes, buds, leaves, husks, barks, flower petals and petal extracts, peels, seed coats and stems. Once the plants and plant parts are obtained, they are cut into small pieces if necessary and then preferably dried into a powder, and they may be referred to herein as “processed plants or plant parts”.

[0042] In the context of plant parts, “derived from” means that the part is either a part produced by the plant and separated or collected (such as seeds, fruits), or an extract of the plant (such as petal extract), or a part removed from the plant.

[0043] For the purpose of interpreting this specification, terms used in the singular form will also include the plural form and vice versa.

[0044] As used herein, unless the context requires otherwise, the terms “comprise / include” (comprise and its variants, such as “comprising”, “comprises” and “comprised”) are not intended to exclude other additives, components, integers or steps.

[0045] When referring to measurable values (such as amounts, lengths of time, etc.), “about” as used herein is intended to cover variations of ±5%, in some cases ±1%, and in some cases ±0.1% from the specified value, as those skilled in the art will understand that such variations are suitable for performing the disclosed methods.

[0046] Throughout the disclosure, aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as the individual values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, and from 3 to 6, as well as the individual numbers within that range, for example 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of how broad the range is.

[0047] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problems or complications and are commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable", i.e., compatible with the other ingredients in the formulation.

[0048] As used herein, "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material included in a composition for purposes other than the pharmacodynamic effect (this does not mean excluding materials that may have certain biological effects). Suitable carriers, excipients, etc. can be found in standard pharmaceutical textbooks, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0049] As used herein, "preventing" or "prevention" is intended to mean at least reducing the likelihood of developing a disease or disorder (or susceptibility thereto) (i.e., at least one clinical symptom of the disease will not develop in an individual who may be exposed to or is susceptible to the disease but has not yet experienced or shown symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known to physicians. Those skilled in the art will understand that "prevention" is not an absolute term. In particularly preferred embodiments, the methods of the invention may be to prevent or reduce the severity of the symptoms of the disease or disorder described herein, or to inhibit or minimize its progression. Accordingly, the methods of the invention can be used for both treatment and prevention.

[0050] The terms "treatment" or "treating" of a subject include delaying, slowing, stabilizing, curing, healing, alleviating, mitigating, altering, remedying, reducing exacerbation, improving, enhancing, or affecting a disease or disorder, the symptoms of a disease or disorder, or the risk (or susceptibility) of a disease or disorder. The term "treatment" refers to any indication of success in the treatment or amelioration of an injury, pathology, or disorder, including any objective or subjective parameter such as alleviation, remission, slowing of the rate of exacerbation; reduction in the severity of a disease; stabilization, reduction of symptoms, or making an injury, pathology, or disorder more tolerable to an individual; slowing the rate of degeneration or decline; and making the endpoint of degeneration less debilitating. Treatment does not necessarily result in the complete elimination of a disease or disorder but can reduce or minimize the complications and side effects of infection and the progression of a disease or disorder. Whether treatment is successful can be monitored by means such as a personal physical examination, CT scan, MRI, or blood biomarkers.

[0051] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound, or a material, composition, or dosage form containing the active compound, or a treatment regimen and its components, which, when administered according to a desired treatment regimen, is effective to produce certain desired therapeutic effects and is commensurate with a reasonable benefit / risk ratio.

[0052] A subject can be a eukaryote, an animal, a vertebrate, a mammal, a rodent (such as a guinea pig, hamster, rat, mouse), a murine (such as a mouse), a canine (such as a dog), a feline (such as a cat), an equine (such as a horse), a primate, an ape (such as a monkey or an ape), a monkey (such as a marmoset, baboon), an ape (such as a gorilla, chimpanzee, orangutan, gibbon), or a human. Detailed Description

[0053] The present application aims to provide another polyherbal formulation (PHF) for the holistic treatment of liver diseases, liver injury, and / or hepatotoxicity, which uses a combination of ingredients that were not all previously known to have any effect or action on these conditions. For example, the PHF of the present invention includes plants of the Apocynaceae family, particularly Rauwolfia serpentina, which has historically been used to treat sleep disorders and insomnia. The PHF of the present invention also seeks to be highly effective, easy to prepare, and have reduced side effects compared to existing PHFs.

[0054] The present invention not only provides a PHF with unexpected therapeutic effects because it includes medicinal plants that do not have known positive effects on the treatment of liver diseases, liver injury, and / or hepatotoxicity, but the PHF preferably has a synergistic effect.

[0055] The PHF according to the present application comprises three medicinal plants from the Apocynaceae, Ecliptaceae, and Phyllanthaceae families or consists of three medicinal plants from the Apocynaceae, Ecliptaceae, and Phyllanthaceae families. The three medicinal plants are especially Rauvolfia serpentina, Eclipta prostrata, and Phyllanthus amarus. The plant Rauvolfia serpentina belongs to the Apocynaceae family, Eclipta prostrata belongs to the Asteraceae family, and Phyllanthus amarus belongs to the Phyllanthaceae family. In a preferred embodiment, although the PHF may contain other components and active ingredients, it only contains plants or plant parts from the Apocynaceae, Ecliptaceae, and Phyllanthaceae families.

[0056] To prepare the drug or medicament according to the present application, if necessary, the plants or plant parts are cut into smaller pieces and then preferably dried into processed plants or plant parts. In a further preferred embodiment, the plant parts are also sterilized. Sterilization can be easily carried out by conventionally known methods, but preferably by pasteurization using microwave and / or ultraviolet irradiation.

[0057] In the PHF of the present invention, the whole plant or any part of the plant can be used, including one or a combination of fruits, seeds, rhizomes, buds, leaves, husks, barks, flower petals and petal extracts, peels, seed coats, and stems. The collected plant parts are preferably washed, cut into small pieces, dried, and crushed or ground into powder form by conventional methods. Then, the powdered plants and plant parts can be used in the PHF preparation.

[0058] Therefore, a multi-herbal preparation is provided, which comprises the following components or consists of the following components:

[0059] a) A plant or plant part of the Ecliptaceae family, especially a plant or plant part of Eclipta prostrata, with a content of at least 30%, preferably at least 35% or 40%; and

[0060] b) A plant or plant part of the Phyllanthaceae family, especially a plant or plant part of Phyllanthus amarus, with a content of at least 15%, preferably at least 20%, and at most 40%; and

[0061] c) A plant or plant part of the Apocynaceae family, especially a plant or plant part of Rauvolfia serpentina, with a content of at least 25%, preferably at least 27%.

[0062] Preferably, the PHF comprises:

[0063] Not more than 70%, preferably not more than 60% of the plant or plant part of the Ecliptaceae family;

[0064] Not more than 30% of the plant or plant part of the Phyllanthaceae family; and

[0065] Not more than 36% of plants or plant parts of the Apocynaceae family.

[0066] Even more preferably, the PHF comprises 43% to 46% of plants or plant parts of the Ecliptaceae family, 23% to 26% of plants or plant parts of the Phyllanthaceae family, and 29% to 32% of plants or plant parts of the Apocynaceae family. For a 100 mg PHF sample of this embodiment, it preferably comprises 44.5 mg to 44.85 mg of plants or plant parts of the Ecliptaceae family, 24.5 mg to 24.6 mg of plants or plant parts of the Phyllanthaceae family, and 30.6 mg to 30.7 mg of plants or plant parts of the Apocynaceae family.

[0067] The plants or plant parts of the PHF are most preferably plants and plant parts from Eclipta prostrata, Phyllanthus niruri, and Rauwolfia serpentine. Accordingly, it can be the whole plant used in the PHF of the present invention, or a part of the whole plant, such as a branch or stem of the plant, or a part of the plant, such as a fruit, seed, rhizome, bud, leaf, shell, bark, petal and petal extract, pericarp and seed coat.

[0068] The plants and plant parts can be obtained by any known method. Then, they are preferably dried by known drying methods and powdered by known methods to provide the processed plant / plant part as referred to herein.

[0069] The pH of the PHF is preferably 4.5 to 6.5 (more preferably 4.5 to 5). In addition, the PHF according to the present application is dissolved in a solvent. Preferred solvents include water, chloroform, ethanol, and ether. For human preparations, the PHF is prepared with pharmaceutically acceptable excipients.

[0070] In addition to the plants and plant parts in the PHF, the PHF may also include other non-plant-derived active ingredients, secondary metabolites selected from one or more of flavonoids, saponins, commestans, phenols, alkaloids, tannins, and lignans.

[0071] The PHF can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. Such methods include the step of combining the processed plants or plant parts from the Apocynaceae, Ecliptaceae, and Phyllanthaceae families with a carrier composed of one or more auxiliary ingredients. Generally, the preparation is made by uniformly and tightly combining the processed plant / plant part with a liquid carrier or a finely divided solid carrier or both.

[0072] The form of the preparation can be liquid, solution, suspension, emulsion, elixir, syrup, tablet, lozenge, granule, powder, capsule, cachet, pill, ampoule, suppository, vaginal suppository, ointment, gel, paste, cream, spray, mixture, foam, lotion, oil, bolus, electuary or aerosol.

[0073] Preparations suitable for oral administration (e.g., by ingestion) can be presented in the following dosage forms: discrete units such as capsules, cachets or tablets, each containing a predetermined amount of tartrate; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; or oil-in-water liquid emulsions or water-in-oil liquid emulsions; boluses; electuaries; or pastes.

[0074] Use of PHF

[0075] It is expected that the PHF of the present invention will be useful for treating liver diseases, liver injury or hepatotoxicity. The various disease spectra constituted by liver diseases include viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), cirrhosis and liver cancer.

[0076] Viral hepatitis can be caused by any known virus that causes inflammation of liver hepatocytes, but commonly it is hepatitis A virus, hepatitis B virus or hepatitis C virus.

[0077] The emergence of non-alcoholic fatty liver disease (NAFLD) as a global health problem further highlights the gap in current treatment methods. Although lifestyle changes such as diet and exercise are the main recommendations, targeted drug interventions are significantly lacking.

[0078] In particular, on the one hand, the PHF of the present invention is expected to provide another option for treating non-alcoholic fatty liver disease (NAFLD), which is a disease continuum characterized by excessive lipid accumulation in hepatocytes. It progresses from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH), and in more severe cases, to liver fibrosis and cirrhosis.

[0079] On the other hand, the PHF of the present invention is also expected to provide another option for treating fatty liver disease (the most common chronic liver disease in Western societies). Fatty liver disease is not caused by alcohol or hepatitis virus, but by the accumulation of fatty acids in the form of triglycerides in hepatocytes of the liver parenchyma. In particular, histological fat accumulation in the liver is a characteristic of non-alcoholic fatty liver disease, manifested by various symptoms, including simple steatosis, steatohepatitis and cirrhosis. In addition, fatty liver disease increases insulin resistance, thereby promoting the breakdown of adipose tissue. The resulting increased supply of liver fatty acids promotes fatty acid oxidation, thereby inducing fat accumulation in hepatocytes.

[0080] In another aspect, the PHF of the present invention is expected to provide another option for providing protection against these processes. The content of MDA produced by lipid peroxidation in the liver was evaluated. Lipid peroxidation can directly damage hepatocytes, exacerbate liver inflammation and mediate fibrogenesis.

[0081] In another aspect, the PHF of the present invention is expected to provide another option for the treatment of hepatic steatosis, which is a hallmark feature of NAFLD, whereby lipid droplets accumulate in hepatocytes in the form of triglycerides. When the accumulation occurs in >5% of hepatocytes, NAFLD is diagnosed histologically.

[0082] In addition to the above, in another aspect, the PHF of the present invention is expected to be an alternative therapy for the following conditions. The triglycerides in the liver with NAFLD are derived from the esterification of glycerol and free fatty acids (FFAs). Triglyceride accumulation occurs when the rate of input or synthesis of FFAs by hepatocytes exceeds the rate of output or catabolism. Obesity, especially insulin resistance (IR), is closely associated with the development of NAFLD. Overexpression of tumor necrosis factor (TNF)-α activates IκB kinase β, which plays an important role in the development of IR by inhibiting the phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2. IR leads to an increase in liver triglyceride levels and ultimately hepatic steatosis through various mechanisms. Insulin fails to inhibit lipolysis in adipose tissue through hormone-sensitive lipase, resulting in an increase in FFAs flowing into the bloodstream, which are then taken up by the liver.

[0083] In another aspect, the PHF of the present invention is expected to be beneficial for the metabolic alterations in lipid profiles and glucose homeostasis and the functional and structural changes of hepatocytes in high-fat diet-induced NAFLD.

[0084] In another aspect, the PHF of the present invention is expected to be an alternative therapeutic agent with hepatitis virus inhibitory activity, preferably with hepatitis A, B or C virus inhibitory activity, most preferably with hepatitis B virus (HBV) inhibitory activity, and is used for the treatment of liver fibrosis, cirrhosis and NAFLD.

[0085] Accordingly, there is provided the use of a multi-herbal preparation in the manufacture of a medicament for treating liver diseases, liver injury or liver toxicity in a subject in need thereof, wherein the medicament comprises or consists of the following ingredients:

[0086] a) a plant or plant part of the Eclipta family, especially a plant or plant part of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0087] b) Plants or plant parts of the Phyllanthaceae family, especially plants or plant parts of Phyllanthus amarus, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0088] c) Plants or plant parts of the Apocynaceae family, especially plants or plant parts of Rauvolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0089] Another aspect of the present invention provides a method for treating liver diseases, liver injuries or liver toxicities in a subject in need thereof, the method comprising administering a multi-herbal preparation comprising or consisting of the following ingredients:

[0090] a) Plants or plant parts of the Ecliptaceae family, especially plants or plant parts of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0091] b) Plants or plant parts of the Phyllanthaceae family, especially plants or plant parts of Phyllanthus amarus, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0092] c) Plants or plant parts of the Apocynaceae family, especially plants or plant parts of Rauvolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0093] Another aspect of the present invention provides a multi-herbal preparation for treating liver diseases, liver injuries or liver toxicities in a subject in need thereof, which comprises or consists of the following ingredients:

[0094] a) Plants or plant parts of the Ecliptaceae family, especially plants or plant parts of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0095] b) Plants or plant parts of the Phyllanthaceae family, especially plants or plant parts of Phyllanthus amarus, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0096] c) Plants or plant parts of the Apocynaceae family, especially plants or plant parts of Rauvolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0097] In each aspect related to treating liver diseases, liver injuries or liver toxicities in a subject in need thereof, the PHF preferably comprises 43% to 46% of plants or plant parts of the Ecliptaceae family, 23% to 26% of plants or plant parts of the Phyllanthaceae family, and 29% to 32% of plants or plant parts of the Apocynaceae family.

[0098] In embodiments of each of the above aspects of the use of the present invention, the subject is infected with a virus that can cause hepatitis, preferably selected from hepatitis A virus, hepatitis B virus, and hepatitis C virus; most preferably hepatitis B virus (HBV). The HBV life cycle includes key stages, mainly the attachment and replication of viral DNA, and the final stage is related to hepatocyte damage. HBV induces liver inflammation, leading to potential long-term consequences, including the development from inflammation to fibrosis, and in severe cases, cirrhosis, thus having a negative impact on liver function.

[0099] Therefore, a comprehensive approach to combating HBV involves adopting a strategy that addresses viral replication and entry, with a focus on eliminating the virus while reducing liver damage. The core of this approach is to reduce inflammation and promote hepatocyte recovery. To achieve this overall strategy, three components of the PHF of the present invention were selected because of their unique properties in controlling HBV infection and promoting hepatocyte repair.

[0100] Accordingly, there is provided the use of a multi-herbal preparation in the manufacture of a medicament for treating a hepatitis virus infection in a subject in need thereof, wherein the multi-herbal preparation of the medicament comprises or consists of the following components:

[0101] a) a plant or plant part of the Ecliptaceae family, particularly a plant or plant part of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0102] b) a plant or plant part of the Phyllanthaceae family, particularly a plant or plant part of Phyllanthus niruri, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0103] c) a plant or plant part of the Apocynaceae family, particularly a plant or plant part of Rauvolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0104] Another aspect of the present invention provides a multi-herbal preparation for treating a hepatitis virus infection in a subject in need thereof, which comprises or consists of the following components:

[0105] a) a plant or plant part of the Ecliptaceae family, particularly a plant or plant part of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0106] b) a plant or plant part of the Phyllanthaceae family, particularly a plant or plant part of Phyllanthus niruri, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0107] c) a plant or plant part of the Apocynaceae family, particularly a plant or plant part of Rauvolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0108] The hepatitis virus is preferably selected from hepatitis A virus, hepatitis B virus, and hepatitis C virus; most preferably hepatitis B virus (HBV).

[0109] The present disclosure should in no way be limited to the exemplary embodiments, examples, and techniques shown in the figures and described below.

[0110] Examples

[0111] The present invention will now be described with reference to the following non-limiting examples. Many different plants and combinations were tested, but the PHF of Rauwolfia SPP., Eclipta SPP., and Phyllanthus SPP. showed the most promising activities against the liver disease spectrum, including synergistic effects.

[0112] In vitro studies evaluated individual plants, but their efficacy was found to be limited. In addition, if only two ingredients were used, the efficacy of this combination was low.

[0113] Physical properties of PHF

[0114] Moisture content

[0115] The moisture content of PHF was measured by placing different masses of PHF powder into a precisely weighed moisture pan. To estimate the drying loss, it was dried in a hot air oven at 105 °C for 3 hours, cooled in a desiccator for 30 minutes, and then immediately weighed. The weight loss was calculated as the content (percentage) of the air-dried material.

[0116] pH determination

[0117] The pH of PHF was determined by preparing stock solutions containing different percentages of PHF. Stock solutions were prepared by dissolving the required mass of PHF in 100 mL of distilled water. The pH of these stock solutions was measured using a standard simple glass electrode pH meter.

[0118] Phytochemical analysis

[0119] Total alkaloid content

[0120] PHF powder (25, 50, and 100 mg) was added to a separating funnel along with 5 mL of PBS (pH 4.7), 5 mL of 0.1% bromocresol green solution, and 4 mL of chloroform. Once the BCG-chloroform complex was formed, (1, 2, 3, and 4) mL of chloroform was successively added and shaken vigorously to extract the complex. The extract was collected in a 10 mL volumetric flask and made up to the mark with chloroform. The absorbance of the complex in chloroform was measured at 470 nm against a blank using a visible spectrophotometer. Atropine standard solutions (0.5, 1.0, 1.5, 2.0, and 2.5) mg / mL were prepared following the above procedure. An atropine calibration curve was plotted for the absorbance at 470 nm by spectrophotometry. The total alkaloid content (TAC) was determined by the following formula and expressed as milligrams of atropine equivalents (AE) per gram of crude extract (John et al., 2015).

[0121]

[0122] Where C is the concentration result obtained from the calibration curve, V is the volume of the crude extract stock solution, and m is the weight of the PHF powder used.

[0123] Total flavonoid content

[0124] A rutin stock solution (1 mg / mL) was prepared by dissolving 100 mg of rutin in 100 mL of methanol and then diluted to rutin standard solutions of (0.02, 0.04, 0.06, 0.08, and 0.10) mg / mL. 0.1 mL of 20% methanol aluminum chloride and one drop of acetic acid were added to each standard solution. The final volume (5 mL) was made up with CH 3 OH. After 40 minutes of incubation, the absorbance of the rutin standard was measured at 415 nm using a visible spectrophotometer. Subsequently, the same analytical reagents were added to PHF powder (25, 50, and 100 mg). After 40 minutes, the absorbance at 415 nm was measured for each concentration. Then the total flavonoid content (TFC) was calculated by the following formula and expressed as milligrams of rutin equivalents (RE) per gram of crude extract (Rao et al., 2016).

[0125]

[0126] Where C is the concentration result obtained from the calibration curve, V is the volume of the crude extract stock solution, and m is the weight of the PHF powder used.

[0127] Total phenolic content

[0128] 1 mL of Folin-Ciocalteu reagent and 2 mL of 7.5% sodium carbonate were added to PHF powder (25, 50, and 100 mg). By adding dH 2Make the total volume reach 7 mL and incubate for 2 hours in the dark at room temperature. Measure the absorbance at 760 nm using a visible spectrophotometer. Prepare gallic acid standard solutions (0.02, 0.04, 0.06, 0.08, 0.10 mg / mL) with the same analytical reagents as above, plot the gallic acid standard curve against the blank and measure the corresponding absorbance at 760 nm. The total phenolic content (TPhC) is calculated by the following formula and expressed as milligrams of gallic acid equivalents (GAE) per gram of crude extract (Shamsa et al., 2008).

[0129]

[0130] Where C is the concentration result obtained from the calibration curve, V is the volume of the stock solution, and m is the weight of the PHF powder used.

[0131] Total tannin content

[0132] Add 500 μL of FCR to PHF powder (25, 50, and 100 mg), then add 1 mL of 7.5% Na 2 CO 3 solution and 8 mL of dH 2 O. Let the reaction mixture stand at room temperature for 30 minutes. Obtain the supernatant by centrifugation and measure the absorbance at 725 nm. Prepare tannic acid standard solutions with concentrations of (0.02, 0.04, 0.06, 0.08, 0.10, 0.2, 0.4, 0.6, 0.8, and 1.0) mg / mL with the same analytical reagents as above, plot the tannic acid standard curve against the blank and measure the corresponding absorbance at 725 nm. The total tannin content (TTC) is calculated by the following formula and expressed as milligrams of tannic acid equivalents (TE) per gram of crude extract (Frederick and V. Mani, 2016).

[0133]

[0134] Where C is the concentration result obtained from the calibration curve, V is the volume of the stock solution, and m is the weight of the PHF powder used.

[0135] Total protein content

[0136] To 2 mL of alkaline copper sulfate reagent, add PHF powder (25, 50, and 100 mg), mix well with a magnetic stirrer at 300 rpm, and incubate at room temperature for 10 minutes. After that, add 0.2 mL of FCR and then incubate for another 30 minutes at room temperature. Measure the absorbance of the crude stem extract relative to the blank at 660 nm using a visible spectrophotometer. Prepare bovine serum albumin standard solutions (0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL) in the same way and plot the BSA standard curve at 660 nm. The total protein content (TPC) is calculated by the following formula and expressed as mg of BSA equivalent (BE) per gram of crude extract (Prabhavathi et al., 2016).

[0137]

[0138] Where C is the concentration result obtained from the calibration curve, V is the volume of the stock solution, and m is the weight of the PHF powder used.

[0139] Total saponin content

[0140] Dissolve PHF powder (25, 50, and 100 mg) in 10 and 20 mL of 20% ethanol, respectively. Then, heat the mixture in a water bath at 55 °C with continuous stirring for 4 hours. The residue obtained after vacuum filtration is re-extracted with 10 and 20 mL of 20% C 2 H 5 OH and reduced to 4 and 6 mL in a water bath at 80 °C. Mix the concentrated solution vigorously with 2 and 6 mL of diethyl ether in a separating funnel. Add approximately 4 and 8 mL of butanol to the collected aqueous layer. Then wash with 2 and 6 mL of 5% w / v aqueous sodium chloride solution. Heat the entire mixture on a water bath and then dry to a constant weight at 40 °C.

[0141] Cytotoxicity

[0142] Prepare 100 mg of PHF liver composition using a combination of 44.75 mg of Eclipta prostrata, 24.58 mg of Phyllanthus niruri, and 30.66 mg of Rauvolfia serpentina (all in dry state).

[0143] Evaluate the cytotoxicity of PHF powder by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytotoxicity of different concentrations of PHF powder against the A2780 epithelial cell line was determined.

[0144] Resuscitate and thaw the A2780 epithelial cell line; transfer it to a falcon tube; add 3 mL of fetal bovine serum; add 4 mL of complete growth RPMI-1640 (containing 10% FCS), and centrifuge the cells at 1500 g for 6 minutes. Pour out the supernatant; suspend the pellet with 6 mL of 1X Dulbecco's Phosphate Buffered Saline (DPBS) and centrifuge at 1500 g for 6 minutes. After centrifugation, pour off the supernatant and suspend the cells with 4 mL of complete growth RPMI-1640 containing 10% FCS. Seed the cells into a T-25 flask. Add approximately 6 mL of complete growth RPMI-1640 containing 10% FCS and incubate at 37 °C in a 5% CO 2 incubator. Regularly monitor whether the cells form a 100% confluent monolayer.

[0145] Extract preparation

[0146] Dissolve the PHF powder in dimethyl sulfoxide to prepare a PHF stock solution. Prepare PHF suspensions of different concentrations by two-fold dilution.

[0147] Cytotoxicity assay

[0148] Seed approximately 200 μL of freshly harvested A2780 epithelial cell line into each well of a 96-well microtiter plate. Then, add 100 μL of complete growth medium to the wells containing the cell line. Incubate the corresponding plate at 37 °C in a humidified incubator containing 5% CO 2 for 24 hours to obtain 100% confluent cells.

[0149] After 24 hours, return the test plate to a sterile state and decant the medium of the epithelial cell line. Then, add 100 μL of each concentration of PHF to the test wells and add 100 μL of complete growth medium to the negative and cell control wells. Incubate the plate at 37 °C in a humidified incubator containing 5% CO 2 for 2 hours. After 2 hours, decant the PHF samples of the epithelial cell line and wash the cells three times with 1X DPBS.

[0150] Under dark conditions, add approximately 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT dye solution) to each well; wrap the plate with aluminum foil and incubate the plate at 37 °C in a humidified incubator containing 5% CO 2 for 4 hours. After incubation, use an inverted phase contrast microscope to photograph the formation of formazan crystals. Subsequently, add 100 μL of solubilization solution to each well. Incubate the plate for 15 minutes and mix the contents of the wells to obtain a uniformly colored solution. Obtain the absorbance value at a wavelength of 570 nm.

[0151] The main structural components of PHF

[0152] The Fourier transform infrared spectroscopy analysis of PHF powder was carried out using an FTIR device. Approximately 0.5 mg of PHF powder and 1 g of anhydrous potassium bromide (KBr) were made into pellets using a hydraulic press. The sample pellets were transferred to a sample holder, and FTIR analysis of the PHF powder was performed.

[0153] In vitro antioxidant activity

[0154] ABTS antioxidant assay

[0155] A working solution was prepared by mixing 1 mL of ABTS solution with 88 mL of 50% ethanol. Various concentrations of PHF powder were prepared by two-fold dilution. L-ascorbic acid was used as a positive control.

[0156] The positive control was prepared by adding 250 μL of L-ascorbic acid at each concentration and 1250 μL of the ABTS working solution. The negative control was prepared by adding 250 μL of methanol and 1250 μL of the ABTS working solution. The test samples were prepared by adding 1250 μL of the ABTS working solution to 250 μL of PHF suspensions at different concentrations and left to stand at room temperature for 4 minutes. The absorbance at 734 nm was read using a visible spectrophotometer. The results were expressed as ascorbic acid equivalents used as a standard. The percentage of free radical scavenging activity of PHF powder and ascorbic acid was calculated by the following formula:

[0157]

[0158] where Ac is the absorbance of the control and As is the absorbance of the test sample. The percentage of ABTS free radical scavenging activity was plotted and the IC 50 .

[0159] DPPH antioxidant assay

[0160] 2 mL of 0.3 mM DPPH was added to PHF suspensions at different concentrations. The reaction mixture was incubated at room temperature in the dark. Exactly 30 minutes after incubation, the absorbance at 517 nm was measured using a visible spectrophotometer.

[0161] Ascorbic acid was used as a positive control. 2 mL of 0.3 mM DPPH was added to each ascorbic acid dilution and the absorbance at 517 nm was measured after incubation for 30 minutes under dark conditions. DPPH (0.3 mM) was used as a control, while CH 3 OH was used as a blank. The percentage of free radical scavenging activity of PHF powder and ascorbic acid was calculated. The percentage of DPPH free radical scavenging activity was plotted and the IC 50 .

[0162] Immunomodulatory effect of PHF

[0163] Freshly isolated peripheral blood mononuclear cells were seeded into 12-well culture plates (2×10 6 cells / well). Different concentrations of PHF liver extract were used, and PHF-treated cells were used as positive controls. Untreated cells were used as negative controls. Supernatants were collected at 24, 48, and 72 hours after stimulation. IL2, IL4, and IFNγ cytokines in the supernatants were screened.

[0164] Anti-HBV activity

[0165] In silico anti-HBV activity

[0166] Protein preparation

[0167] The required protein sequences were initially downloaded from Genbank (NCBI) and protein blast was performed. Then sequences with high similarity indices were considered and used for protein modeling. The uniprot database was used to analyze different conformations of the same protein and used for homology protein modeling. The Swiss Model was used to model proteins of different conformations, and the model was analyzed using the structure evaluation tool in the Swiss Bioinformatics database. Conformations with high Ramachandran plot values and low clash scores were selected for further processing. The Qmean value of the model was also estimated to analyze the integrity of the developed model (homology protein structure).

[0168] Table 1 shows the protein modeling of different conformations analyzed using the structure evaluation tool in the Swiss Bioinformatics database.

[0169] Table 1

[0170]

[0171] Selected conformer: Hepatitis B virus polymerase-1

[0172] Preparation of ligand and target protein

[0173] The generated protein model was used for the docking process. The pymol software was used to analyze the errors in the three-dimensional protein structure. Similarly, the pymol visualization tool was also used to remove ions, molecules, and standard inhibitors (if present).

[0174] Anti-HBsAg of PHF

[0175] Equal volumes of HBsAg-positive plasma and PHF were mixed and incubated at 37 °C for 5 days. The mixture was assayed daily for 5 days using a commercial HBsAg ELISA kit. Plasma tubes containing only solvent were used as controls. The binding effect of the extract was analyzed daily.

[0176] In vivo toxicity study

[0177] In vivo assessment of the acute toxicity of the test substance in mice

[0178] Observe the signs of toxicity in the animals, including changes in the skin and hair, eyes and mucous membranes, as well as changes in the respiratory, circulatory and behavioral patterns. Pay attention to observe tremors, convulsions, salivation, diarrhea, lethargy, sleep. Dissect the internal organs (brain, heart, lungs, spleen, stomach, kidneys, liver), remove the fat, weigh them, and conduct macroscopic observations.

[0179] In vivo assessment of the subacute toxicity of the test substance in mice

[0180] According to OECD guideline 407, a subacute oral toxicity study of PHF was conducted using female Swiss albino mice. A total of 18 female mice were used in the study. The mice were randomly divided into 3 groups of 6 each, and the amount of PHF administered per unit body weight to each group of mice in the three groups was 100 mg / kg, 200 mg / kg, and 400 mg / kg, respectively.

[0181] Experimental design of subacute toxicity study

[0182] Table 2 shows the experimental design of the three groups.

[0183] Table 2

[0184] Group - I Oral administration of PHF at 100 mg / kg for 60 days (n = 6) Group - II Oral administration of PHF at 200 mg / kg for 60 days (n = 6) Group - III Oral administration of PHF at 400 mg / kg for 60 days (n = 6)

[0185] Experimental protocol

[0186] Prepare a suspension of PHF and Tween 80. Tween 80 is a polysorbate, which is a non-ionic surfactant and emulsifier, a synthetic compound in the form of a viscous water-soluble yellow liquid. Adjust the volume with distilled water and administer it to the mice in 3 different groups daily for 60 consecutive days, with each group receiving a different dose, namely 100 mg / kg, 200 mg / kg, and 400 mg / kg. The mice in all groups were fed normal food and water under the same conditions. Observe their mortality, signs of toxicity and behavioral changes. Since the start of the study, changes in body weight and food intake have also been recorded. On day 0 and day 60, the mice were weighed, urine samples and blood samples were collected for various biochemical analyses, and the mice were sacrificed by overdose inhalation of anesthetic ether, and then histopathological studies were conducted.

[0187] Biochemical parameters and hematology

[0188] On day 0 and day 60, urine samples and blood samples were collected from the mice in all groups. The blood was used for hematological studies such as red blood cell count (RBC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, white blood cell count (WBC), lymphocytes, monocytes, eosinophils and neutrophils, and the serum and urine samples were used to analyze various biochemical parameters such as alkaline phosphatase (ALP), serum urea, serum glucose, serum cholesterol, serum total protein, serum total bilirubin, serum aspartate aminotransferase (AST), serum creatinine.

[0189] Histopathological study

[0190] Autopsy and gross examination were performed on the internal organs (liver, heart, kidney, uterus, brain, spleen, lung, stomach, ovary, pancreas, trachea, ileum, esophagus, large intestine, aorta, bone and skin). Then, the above internal organs were dissected, the fat was removed, weighed and macroscopically observed. Finally, they were fixed in 10% buffered formalin solution for histopathological examination.

[0191] In vivo evaluation of the effect of the test substance on liver fibrosis in rats

[0192] Table 3 shows the experimental design of the six groups.

[0193] Table 3

[0194] Group - I Normal rats orally administered with vehicle and normal diet pellets (n = 6) Group - II Control group orally administered with vehicle and paracetamol (1 gm / kg) (n = 6) Group - III Standard silymarin (200 mg / kg) and paracetamol (1 gm / kg) (n = 6) Group - IV Paracetamol (1 gm / kg) and PHF (100 mg / kg) (n = 6) Group - V Paracetamol (1 gm / kg) and PHF (200 mg / kg) (n = 6) Group - VI Paracetamol (1 gm / kg) and PHF (400 mg / kg) (n = 6)

[0195] Treatment protocol

[0196] The animals were divided into 6 groups of 6 animals each. Except for the normal group, all animals were given paracetamol (1 gm / kg, orally) for 7 consecutive days.

[0197] Group I received only the vehicle for 7 consecutive days as a normal control. Group II served as a negative control and received paracetamol (1 gm / kg, orally) for the first 7 days. Group III served as a positive control and received paracetamol and silymarin (200 mg / kg, orally) for the first 7 days. Group IV received paracetamol (1 gm / kg, orally) for the first 7 days and a lower dose of PHF (100 mg / kg) from day 0 to day 12. Group V received paracetamol (1 gm / kg, orally) for the first 7 days and a medium dose of PHF (200 mg / kg) from day 0 to day 12. Group VI received paracetamol (1 gm / kg, orally) for the first 7 days and a higher dose of PHF (400 mg / kg) from day 0 to day 12.

[0198] Blood biochemical analysis

[0199] On the 13th day, blood samples were collected from the rats in all groups. Serum was separated and used for the analysis of various biochemical parameters, such as alkaline phosphatase (ALP), total serum protein, total serum bilirubin, serum AST, serum alanine aminotransferase (ALT), globulin, albumin, and γ-glutamyl transferase (GGT).

[0200] Histopathological study

[0201] The livers were collected in 10% formalin for proper fixation. The tissues were processed and embedded in paraffin. Sections with a thickness of 5-6 μm were cut and stained with hematoxylin and eosin dyes, and histopathological changes were observed under a microscope.

[0202] Liver RT-PCR gene expression detection

[0203] The livers were collected and used for gene expression detection, such as the detection of collagen 1α1, tissue inhibitor of metalloproteinase-1, and transforming growth factor β.

[0204] In vivo evaluation of the effect of the test substance on rat liver cirrhosis

[0205] Table 4 shows the experimental design of the six groups.

[0206] Table 4

[0207] Group - I Normal rats orally administered with vehicle and normal diet pellets (n = 6) Group - II <![CDATA[The control group was orally administered the vehicle and intraperitoneally injected with 8% carbon tetrachloride (CCl 4 )(1 ml / kg) (n = 6)]]> Group - III <![CDATA[Silymarin standard (200 mg / kg) and intraperitoneal injection of 8% CCl 4 (1 ml / kg) (n = 6)]]> Group - IV <![CDATA[Intraperitoneal injection of 8% CCl 4 (1 ml / kg) and PHF (100 mg / kg) (n = 6)]]> Group - V <![CDATA[Intraperitoneal injection of 8% CCl 4 (1 ml / kg) and PHF (200 mg / kg) (n = 6)]]> Group - VI <![CDATA[Intraperitoneal injection of 8% CCl 4 (1 ml / kg) and PHF (400 mg / kg) (n = 6)]]>

[0208] Treatment protocol

[0209] The animals were divided into six groups of 6 rats each. Except for the normal control, all animals were anesthetized with CCl 4 (1 ml / kg, oral 8%) twice a week (on Monday and Thursday) for 8 weeks. Group I received only the vehicle 4 times a week (on Tuesday, Wednesday, Friday, and Saturday) and served as the normal control. Group II served as the negative control and received CCl 4 (1 ml / kg, oral 8%) twice a week (on Monday and Thursday) for 8 weeks. Group III served as the positive control and received silymarin (0.2 gm / kg) 4 times a week (on Tuesday, Wednesday, Friday, and Saturday) for 8 weeks. Group IV received a lower dose treatment, Group V received a medium dose treatment, and Group VI received a higher dose treatment, 4 times a week (on Tuesday, Wednesday, Friday, and Saturday) for 8 weeks. Groups III to VI were treated with the same amount of CCl 4 as Group II.

[0210] Blood biochemical analysis

[0211] On days 0, 28, and 56, blood samples were collected from rats in all groups. Serum was separated and used for analysis of various biochemical parameters, such as alkaline phosphatase (ALP), total serum protein, total serum bilirubin, serum AST, serum ALT, globulin, albumin, and γ-glutamyl transferase (GGT).

[0212] Histopathological study

[0213] The livers were collected in 10% formalin for proper fixation. The tissues were processed and embedded in paraffin. Sections with a thickness of 5 - 6 μm were cut, stained with hematoxylin and eosin dyes, and histopathological changes were observed under a microscope.

[0214] Liver RT-PCR gene expression detection

[0215] Livers were collected and used for gene expression detection, such as detection of P53, TGFβ1, TIMP1, MMP2, and Collα.

[0216] Results and discussion

[0217] MTT assays were performed on A2780 epithelial cells and THP-1 phagocytic cells. The A2780 epithelial cell line is a human ovarian cancer cell line established from tumor tissues of untreated patients with ovarian endometrioid adenocarcinoma and was used for toxicity testing.

[0218] Physical properties

[0219] Moisture content

[0220] Table 5 shows the moisture content in different quality PHFs.

[0221] Table 5

[0222]

[0223] pH

[0224] Table 6 shows the pH of PHFs at different percentages.

[0225] Table 6

[0226]

[0227]

[0228] Heavy metal analysis

[0229] Table 7 shows the heavy metal screening of PHF under various test parameters.

[0230] Table 7

[0231]

[0232] Solubility of Cellulose Fibers

[0233] Table 8 shows the solubility of PHF in various solvents.

[0234] Table 8

[0235]

[0236] Phytochemical Analysis

[0237] Table 9 shows the phytochemical contents of PHF in different samples.

[0238] Table 9

[0239]

[0240] Note: Flavonoids - rutin equivalent; Total phenols - gallic acid equivalent; Tannins - tannic acid equivalent, and Total protein - BSA equivalent. Data are expressed as mean ± standard error. *p < 0.05.

[0241] In Vitro Antioxidant Activity

[0242] Table 10 shows the amounts of antioxidants present in PHF of different samples.

[0243] Table 10

[0244]

[0245] The antioxidant activity of PHF powder was evaluated using DPPH and ABTS radical scavenging assays. The ABTS assay is based on the generation of blue / green ABTS·+ which can be reduced by antioxidants; while the DPPH assay is based on the reduction of purple DPPH· to 1,1-diphenyl-2-picrylhydrazine. Both assays indicated that PHF is a potent antioxidant and the antioxidant activity of PHF increased significantly when the concentration of PHF samples increased. PHF may exhibit antioxidant properties due to the presence of secondary metabolites and other major components in it.

[0246] Immunomodulatory Effect of PHF

[0247] Table 11 shows the induction of cytokines by various plant extracts.

[0248] Table 11

[0249]

[0250] Anti-HBV Activity

[0251] In Silico Anti-HBV Activity

[0252] The docking results of hepatitis B virus polymerase with the selected ligands are summarized in Table 12. Binding energy (Kcal / mol), RMSD value, and the hydrogen bonds formed are used to evaluate the binding affinity of the inhibitors.

[0253] Table 12 shows the docking results of the selected ligands with hepatitis B virus polymerase.

[0254] Table 12

[0255]

[0256] The docking results of sodium taurocholate cotransporting polypeptide (ntcp) with the selected ligands are summarized in Table 13 below. Binding energy (Kcal / mol), RMSD value, and the hydrogen bonds formed are used to evaluate the binding affinity of the inhibitors.

[0257] Table 13 shows the docking results of the selected ligands with sodium taurocholate cotransporting polypeptide (ntcp).

[0258] Table 13

[0259]

[0260]

[0261] Virtual screening was performed on fourteen selected phytochemical compounds using molecular docking analysis. Initially, two proteins, namely human hepatitis B virus polymerase and sodium taurocholate cotransporting polypeptide (ntcp), were selected as the targets for this process. The gene sequences of the protein targets were taken from the Uniport web server, and all conformational isomers (different variants of the same sequence) present in the individual sequences were also considered. Then, protein homology modeling was performed on the sequences of different conformational isomers of the same protein.

[0262] Finally, additional charges and torsions were added to the three-dimensional structure file of the ligand and the target protein was added to evaluate the binding affinity, and then the binding efficiency between the target molecules was evaluated. Docking boundaries were created to target the active sites required for the identified proteins. The results were interpreted by comparing the binding energies of different ligands with the minimum inhibition constant and the maximum number of formed bonds. When analyzing the binding energy of the docking results of the ligands with hepatitis B virus polymerase, the binding energy of demethylwedelolactone was the lowest, at -7.9 Kcal / mol; followed by geraniin, at -7.6 Kcal / mol; comestan, at -7.2 Kcal / mol; reserpine and serpentine, at -6.9 Kcal / mol. Similarly, considering the number of hydrogen bonds formed, phyllanthusiol A had three hydrogen bonds, followed by phyllanthusinol B, geraniin, lignan, apigenin, and comestan, all of which formed two hydrogen bonds with hepatitis B virus polymerase.

[0263] Finally, after careful observation and consideration of all parameters including the RMSD value, it was found that both norwedelolide and geraniin were effective against hepatitis B virus polymerase. Similarly, the interaction between the ligands and the target protein sodium taurocholate cotransporting polypeptide (ntcp) was also evaluated, and it was found that comestan showed the highest binding energy of -8.6 Kcal / mol; followed by geraniin at -7.7 Kcal / mol; norwedelolide at -7.6 Kcal / mol; hinokitiol at -7.4 Kcal / mol and apigenin at -6.9 Kcal / mol. When considering the number of hydrogen bonds formed, all 13 ligands formed one hydrogen bond with the target protein ntcp. Finally, it was shown that both comestan and norwedelolide were found to be effective against the target ntcp. Overall, the study yielded a positive result that almost all 13 ligands exhibited low binding energy and good binding affinity for the two target proteins. In particular, phytochemicals such as comestan, geraniin and norwedelolide were found to be highly effective against the two target proteins, namely human hepatitis B virus polymerase and ntcp.

[0264] Anti-HbsAg (in vitro) of PHF

[0265] Table 14 shows the anti-HbsAg activity of PHF at different concentrations.

[0266] Table 14

[0267] PHF concentration (ng / ml) % Inhibition 100 81 50 73 25 54 12.5 39 6.25 31 3.12 23 1.56 19 0 -

[0268] PHF has anti-HbsAg activity against the test virus. As shown in Table 14, it can be seen that PHF at a concentration of 100 ng / mL inhibits 81% of the virus.

[0269] In vivo toxicity study

[0270] Acute toxicity of the test substance in mice was evaluated in vivo

[0271] In the acute toxicity study, oral administration of 2000 mg / kg of PHF to mice did not cause any death. The mice did not show any obvious signs of toxicity such as changes in skin, eyes, hair and mucous membranes. In addition, these mice did not show any behavioral changes including salivation, sleep, coma, lethargy, diarrhea. Throughout the study period, these animals were found to be normal. Histopathological observation of internal organs did not reveal such changes. Therefore, it was found that LD 50 exceeds 2000 mg / kg.

[0272] Spleen

[0273] Examination of H&E stained sections of the spleens of experimental mice showed characteristics of normal spleen tissue structure, such as no parenchymal or capsular fibrosis, no fat deposition, no spleen necrosis, and no formation of vacuoles in splenic histiocytes.

[0274] Stomach

[0275] Examination of H&E stained sections of the stomachs of experimental mice showed characteristics of normal stomach tissue structure without pathological changes, such as no ulcers, no congestion, no bleeding, and no epithelial cell degeneration and necrosis observed in the gastric mucosa layer.

[0276] Kidney

[0277] Examination of H&E stained sections of the kidneys of experimental mice showed characteristics of normal kidney tissue structure, such as prominent critical tubules and Bowman's capsules, normal glomerular structure, no renal tubular damage, no bleeding, no increased neutrophil infiltration, no tubule dilation, no glomerular atrophy, and no interruption of the basement membrane around necrotic tubules.

[0278] In vivo assessment of the subacute toxicity of the test substance in mice

[0279] Effect of different doses of PHF on body weight and food intake

[0280] Table 15 shows the effect of different doses of PHF on body weight and food intake; there were no changes in body weight and food intake compared to day 0.

[0281] Table 15

[0282]

[0283] Effect of different doses of PHF on serum biochemical parameters

[0284] Tables 16 and 17 show the effect of different doses of PHF on serum biochemical parameters.

[0285] Tables 16 and 17 confirmed that there were significant increases (p<0.05 to p<0.001) in serum glucose, cholesterol, total protein, total bilirubin, AST, creatinine, and urea levels compared to day 0. There was no significant increase in ALP level compared to day 0.

[0286] Table 16

[0287]

[0288] Table 17

[0289]

[0290] Effect of different doses of PHF on urinary biochemical parameters

[0291] Table 18 shows the effect of different doses of PHF on urine biochemical parameters. Table 18 confirmed that glucose and total protein were significantly increased (p < 0.05 to p < 0.001) compared to day 0, while total bilirubin and urine pH did not change, and there was no blood and white blood cells in the urine compared to day 0. Each parameter was evaluated as the percentage change from day 0 to day 60.

[0292] Table 18

[0293] Group Glucose Total protein Total bilirubin Urine pH Blood White blood cells PHF 100 mg / kg 40.69 -78.50 1.32 1.97 A* A PHF 200 mg / kg 42.92 -86.22 9.77 0.56 A A PHF 400 mg / kg 35.51 -76.96 0.78 1.68 A A

[0294] *A represents "absent", which means no change (%) from day 0 to day 60.

[0295] Tables 19 and 20 show the effect of different doses of PHF on complete blood count (CBC). They confirmed that hemoglobin (Hb), total count, neutrophils, eosinophils, red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular Hb concentration, platelet count, and packed cell volume were significantly increased (p < 0.05 to p < 0.001) compared to day 0, while lymphocytes were not significantly increased compared to day 0. The results in Tables 19 and 20 show the percentage change from day 0 to day 60.

[0296] Table 19

[0297]

[0298] Table 20

[0299]

[0300] Table 20 confirmed that mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular Hb concentration, platelet count, and packed cell volume were significantly increased compared to day 0.

[0301] Hematological parameter examination can be used to identify any harmful effects of foreign compounds included in polyherbal preparations on the blood. Compared to most other mammals, mice have a lower RBC count and a normal mean MCV of 45 to 55 fL when healthy. According to Table 20, in animals treated with 400 mg / kg of PHF, the MCV count and mean corpuscular hemoglobin (pg) increased, but this increase was within the normal range.

[0302] The results of the subacute toxicity study on mice showed that PHF at 100 mg / kg, 200 mg / kg, and 400 mg / kg did not cause any signs of toxicity or death in the animals. Treatment with PHF did not result in any significant changes in animal body weight and food intake. Hematological parameters were used to study the degree of toxicity of PHF. In this study, female mice were administered PHF for 60 days, and all blood parameters changed significantly except for lymphocytes.

[0303] The function of the liver was evaluated through serum. A decrease in the serum levels of total protein, bilirubin, and albumin indicates a decline in function, which is obvious in liver injury or liver diseases. The serum parameters and liver histology of female mice did not change at all before and after treatment. Therefore, it can be concluded that the use of PHF is safe.

[0304] Effect of different doses of PHF on histopathological changes

[0305] Examination of H&E stained sections of the internal organs (liver, heart, kidney, uterus, brain, spleen, lung, stomach, ovary, pancreas, trachea, ileum, esophagus, large intestine, aorta, bone, and skin) of experimental mice showed the characteristics of the tissue structure, and there were no changes in its normal histopathological structure.

[0306] In vivo evaluation of the effect of the test substance on liver fibrosis in rats

[0307] Effect of PHF on serum biochemical parameters

[0308] Tables 21 and 22 list the percentage change data from Day 0 to Day 12.

[0309] Table 21 confirmed that compared with the control group, the serum ALP and SGOT in the treatment group decreased significantly (p < 0.05 to p < 0.001), while the serum albumin in the treatment group increased significantly compared with the control group (p < 0.05).

[0310] Table 22 confirmed that compared with the control group, the serum SGPT and total bilirubin in the treatment group decreased significantly (p < 0.05 to p < 0.001). While the serum total protein and globulin in the treatment group increased significantly compared with the control group (p < 0.05).

[0311] Table 21

[0312] Treatment group ALP Albumin SGOT Normal 146.98 -10.7 470.4 Control 10749.10 -81.7 802.9 Standard 322.97 -16.6 -10.1 T1 (100 mg / kg) 1211.71 -68.7 -22.5 T2 (200 mg / kg) -25.01 -55.3 160.2 T3 (400 mg / kg) -4.54 -35.2 -72.6

[0313] Table 22

[0314]

[0315]

[0316] Effect of PHF on histopathological studies

[0317] Effect of different doses of PHF on paracetamol-induced liver injury in albino rats. Photomicrographs of the liver of different treatment groups stained with hematoxylin and eosin at 40X magnification. The normal group showed constriction of the portal vein, the control group showed dilation of the portal vein, and a completely altered hepatocyte structure, centrilobular necrosis, hepatic steatosis, and fatty changes in the large blood vessels were also observed. However, in the treatments with 100 mg / kg, 200 mg / kg, and 400 mg / kg PHF, the results showed that the structure was basically normal, without centrilobular necrosis and hepatic steatosis.

[0318] ALP is a hydrolyzable enzyme excreted through bile and is present in bile duct cells. Hepatotoxicity leads to biliary congestion, preventing ALP from being excreted from the body, resulting in an increase in ALP levels in the control group. Compared with the control group, the PHF treatment group significantly reduced the ALP levels in a dose-dependent manner.

[0319] The PHF treatment group recovered from the elevated liver markers and decreased protein levels in the blood exuded from hepatocytes.

[0320] In vivo evaluation of the effect of test substances on cirrhosis in rats

[0321] Effect of PHF on serum biochemical parameters

[0322] Table 23 confirmed that compared with the control group, the serum ALP and SGOT in the treatment group were significantly decreased (p < 0.05 to p < 0.001), while the serum albumin in the treatment group was significantly increased compared with the control group (p < 0.05 to p < 0.001).

[0323] Table 23 shows the effect of PHF on serum biochemical parameters.

[0324] Table 23

[0325]

[0326] Table 24 confirmed that compared with the control group, the serum SGPT and total bilirubin in the treatment group were significantly decreased (p < 0.05 to p < 0.001). While the serum total protein in the treatment group was significantly increased compared with the control group (p < 0.05 to p < 0.001).

[0327] Table 24 shows the effect of PHF on serum biochemical parameters.

[0328] Table 24

[0329]

[0330] Table 25 confirmed that, compared with the control group, the serum globulin in the treatment group increased significantly (p < 0.05 to p < 0.001).

[0331] Effect of PHF on histopathological studies

[0332] Effect of different doses of PHF on CCl 4 -induced liver injury in Sprague Dawley rats. Photomicrographs at 40x magnification of the livers of different treatment groups stained with hematoxylin and eosin. The normal group showed constriction of the portal vein, the control group showed dilation of the portal vein, and complete alteration of hepatocyte structure, centrilobular necrosis, hepatic steatosis, and large vessel fatty changes were also observed. However, in the treatments with 100 mg / kg, 200 mg / kg, and 400 mg / kg PHF, the results showed that the structure was basically normal, without centrilobular necrosis and hepatic steatosis.

[0333] Table 25 shows the effect of PHF on serum biochemical parameters.

[0334] Table 25

[0335]

[0336]

[0337] CCl 4 is an exogenous compound and a chemical commonly used to produce experimental liver injury. CCl 4 is a colorless, transparent, volatile liquid used to induce liver injury in experiments.

[0338] As is well known, CCl 4 causes hepatotoxicity through metabolic activation, so it selectively causes hepatocyte toxicity that maintains semi-normal metabolic functions. CCl 4 The tissue injury mechanism begins with the CYP enzyme system forming CCl 3 . CCl 4 is biotransformed by the cytochrome P450 system in the endoplasmic reticulum to produce trichloromethyl radicals (CCl 3 ).

[0339] Trichloromethyl radicals combine with cellular lipids and proteins in the presence of oxygen to form trichloromethylperoxy radicals, which may attack lipids on the endoplasmic reticulum membrane faster than trichloromethyl radicals. Therefore, trichloromethylperoxy radicals cause lipid peroxidation or degrade cell membranes through covalent binding of lipids to proteins, resulting in liver injury.

[0340] Liver injury activates Kupffer cells, leading to the release of early inflammatory mediators such as superoxide anions, which in turn leads to the formation of reactive oxygen species (ROS), especially peroxynitrites and hydrogen peroxide (H 2 O 2 ), resulting in oxidative stress. The disruption of Ca 2+ homeostasis ultimately leads to cell death.

[0341] In this study, it was observed that the administration of CCl 4 significantly decreased the serum total protein level and significantly increased the serum marker enzyme levels. The administration of CCl 4 resulted in a significant increase in the levels of serum AST (SGPT), ALT (SGOT), total bilirubin, and ALP (p < 0.05 to p < 0.001), and a significant decrease in albumin, total protein, and globulin (p < 0.05 to p < 0.001). This may be due to oxidative stress causing liver cell membrane disorders, leading to an increase in the levels of cytoplasmic enzymes such as ALT, AST, and ALP in the serum.

[0342] The research results showed that after liver injury was induced by CCl 4 , the serum levels of these enzymes increased. The study also showed that the administration of PHF could ameliorate the subsequent increase in the levels of AST, ALT, total bilirubin, and ALP caused by the administration of CCl 4 . The levels of ALT, AST, total bilirubin, and ALP decreased significantly (p < 0.05 to p < 0.001), and albumin, total protein, and globulin increased significantly (p < 0.05 to p < 0.001). This is because PHF has strong antioxidant properties and can regulate the integrity of the liver cell membrane, which clearly demonstrates the hepatoprotective effect observed after the administration of PHF.

[0343] In vivo evaluation of the effects of test substances on non-alcoholic fatty liver disease (NAFLD)

[0344] Effects of PHF on serum biochemical parameters

[0345] Tables 26 to 29 show the effects of PHF on serum biochemical parameters. Table 30 shows the effects of PHF on liver tissue biochemical parameters.

[0346] Table 26 confirmed that compared with the control group, the levels of serum ALP, SGOT, and SGPT in the treatment group and the standard group decreased significantly (p < 0.001).

[0347] Table 26

[0348]

[0349] Table 27 confirmed that compared with the control group, the total serum bilirubin, creatinine, and urea in the treatment group and the standard group decreased significantly (p < 0.001).

[0350] Table 27

[0351]

[0352] Table 28 showed that compared with the control group, the serum uric acid, glucose, and cholesterol in the treatment group and the standard group decreased significantly (p < 0.001).

[0353] Table 28

[0354]

[0355] Table 29 confirmed that compared with the control group, the triglyceride and LDL in the treatment group and the standard group decreased significantly (p < 0.001), while compared with the control group, the HDL in the treatment group and the standard group increased significantly (p < 0.001).

[0356] Table 29

[0357]

[0358] Table 30 showed that compared with the control group, the total cholesterol, total protein, malondialdehyde (MDA), and triglyceride in the treatment group and the standard group decreased significantly (p < 0.05 to p < 0.001).

[0359] Table 30

[0360]

[0361]

[0362] Pre-clinical evaluation of the hepatoprotective activity of PHF against NAFLD, liver cirrhosis, fibrosis, and anti-HBV in an experimental model, and evaluation of the acute and subacute toxicity of the polyherbal formulation in animals

[0363] 1. In vivo evaluation of the effect of the test substance on liver fibrosis in rats

[0364] The experiment for evaluating the effect of the test substance on liver fibrosis was modified to confirm the synergistic effect of PHF of the present invention.

[0365] 1. Experimental design

[0366] RA: Plants of the genus Rauwolfia (Rauwolfia spp.), Rauvolfia serpentina

[0367] PA: Plants of the genus Phyllanthus (Phyllanthus spp.), Phyllanthus amarus

[0368] EA: Plants of the genus Eclipta spp., Eclipta prostrata

[0369] Table 31: Treatment Regimen

[0370] Group - I Normal rats orally administered with vehicle and normal diet pellets (n = 6) Group - II Control group orally administered with vehicle and paracetamol (1 gm / kg) (n = 6) Group - III Paracetamol (1 gm / kg) and PHF (200 mg / kg) (n = 6) Group - IV Paracetamol (1 gm / kg) and RA (200 mg / kg) (n = 6) Group - V Paracetamol (1 gm / kg) and PA (200 mg / kg) (n = 6) Group - VI Paracetamol (1 gm / kg) and EA (200 mg / kg) (n = 6)

[0371] Animals and treatments

[0372] Six groups of animals were used in the study, with six animals in each group. Except for the normal control group, all groups received paracetamol (1 g / kg, orally) daily for seven consecutive days.

[0373] · Group I: This group served as the normal control group and received only the vehicle (the substance used to deliver the test compound) for seven consecutive days.

[0374] · Group II: This group served as the negative control group and received paracetamol (1 g / kg, orally) for the first 7 days.

[0375] · Group III: This group received a test dose of PHF (200 mg / kg, orally) from day 0 to day 12.

[0376] · Groups IV, V, and VII: These groups received 200 mg / kg of RA, PA, and EA orally, respectively, from day 0 to day 12.

[0377] Blood Biochemical Analysis

[0378] On day 13, blood samples were collected from the rats in all groups. Serum was isolated from these samples for subsequent analysis of various biochemical parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and platelet count. The AST / ALT ratio is an indicator of the type and degree of liver injury. A ratio greater than 1 may indicate severe liver disease.

[0379] FIB-4 Index: Non-invasive Assessment of Liver Fibrosis

[0380] The FIB-4 index is a non-invasive method for assessing the severity of liver fibrosis. The lower the FIB-4 index value, the lighter the degree of fibrosis. The calculation formula is as follows:

[0381] FIB-4 = Age (years) × AST / Platelet Count × √ALT

[0382] Percentage reduction in FIB-4:

[0383] To evaluate the alleviation of liver fibrosis, the percentage change in the FIB-4 index can be calculated using the following formula:

[0384] Reduction percentage = (FIB-4 control - FIB-4 treatment) / FIB-4 control × 100%

[0385] Evaluation of synergistic interaction

[0386] To determine whether the combination therapy exhibits a synergistic effect, the Bliss independence model (Bliss, C. I. (1939). The toxicity of poisons applied jointly. Annals of Applied Biology, 26(3), 585-615) was adopted. This model assumes that the effects of each treatment are independent of each other.

[0387] Bliss independence model:

[0388] The predicted combined effect (E pred ) of PHF was calculated using the following formula:

[0389] E pred = E 1 + E 2 + E 3 - E 1 * E 2 - E 1 * E 3 - E 2 * E 3 + E 1 * E 2 * E 3

[0390] Where:

[0391] · E pred : The predicted combined effect of PHF

[0392] · E 1 、E 2 and E 3 : The individual effects of RA, PA, and EA, respectively.

[0393] Results

[0394] Table 32: Effects of PHF and RA, PA, EA and AST / ALT ratio

[0395] Treatment group AST (IU / L) ALT (IU / L) Ratio Remarks Normal 45 50 0.90 Healthy liver Control 250 200 1.25 Advanced fibrosis PHF (200 mg / kg) 57 60 0.60 Mild liver disease RA (200 mg / kg) 150 100 2.82 Advanced fibrosis PA (200 mg / kg) 130 110 2.29 Advanced fibrosis EA (200 mg / kg) 130 100 2.69 Advanced fibrosis

[0396] AST / ALT > 1 in advanced fibrosis

[0397] Table 33: Effects of PHF and RA, PA, EA on serum biochemical parameters

[0398]

[0399] Table 34: Summary of the Therapeutic Effects on Liver Function

[0400]

[0401]

[0402] The PHF of the present invention showed the most significant improvement in liver function at a dose of 200 mg / kg, with the highest reduction in the FIB-4 index, indicating effective alleviation of fibrosis. The AST / ALT ratio was 0.60, indicating a protective effect on the liver. Combined with the FIB-4 index, this indicated a significant improvement in liver function and a significant reduction in fibrosis.

[0403] In contrast, the administration of RA alone at a dose of 200 mg / kg was much less effective in reducing liver injury, with a high AST / ALT ratio indicating severe liver injury and a small degree of fibrosis alleviation. The administration of PA alone at a dose of 200 mg / kg had a moderate effect on alleviating liver fibrosis, with a moderate AST / ALT ratio. Similarly, the administration of EA alone at a dose of 200 mg / kg showed some alleviation of liver fibrosis, but there was still obvious liver injury, manifested as a high AST / ALT ratio.

[0404] Confirmation of Synergistic Effect

[0405] The Bliss independence model was used to determine whether the effects of the components (RA, PA, and EA) in PHF were synergistic.

[0406] Expected combined reduction in FIB-4 index:

[0407] Individual effects

[0408] · RA: 15% reduction

[0409] · PA: 40% reduction

[0410] · EA: 30% reduction

[0411] Assuming independent combination of RA, PA, and EA:

[0412] E RA+PA = 0.15 + 0.40 - 0.15 × 0.40 = 0.49

[0413] E RA+PA+EA = 0.49 + 0.30 - 0.49 × 0.30 = 0.693

[0414] Expected reduction in FIB-4 index: 69.3%

[0415] For PHF, a 94% reduction in the FIB-4 index was observed.

[0416] Since 94% > 69.3%, the observed PHF effect was greater than the expected combined effect, indicating the presence of synergy.

[0417] Discussion and Conclusions

[0418] The therapeutic potential of the PHF of the present invention in treating liver fibrosis and cirrhosis has been comprehensively evaluated. Compared with untreated animals, the biochemical parameters in the liver function tests of animals treated with PHF showed a return to normal levels, highlighting the effectiveness of the drug in treating liver diseases, especially fibrosis and cirrhosis. Histopathological analysis of the livers of treated animals showed that their structure was close to normal, characterized by the absence of centrilobular necrosis and hepatic steatosis.

[0419] Notably, the PHF of the present invention also showed synergy.

[0420] Even more notably, consistent results were obtained over different treatment durations, highlighting the adaptability and reliability of PHF. Liver fibrosis responded to PHF treatment within just 7 days, while treating cirrhosis and fatty liver conditions required a longer treatment duration of 56 days. These findings highlight the good efficacy of PHF in alleviating liver fibrosis, cirrhosis, and fatty liver, providing potential therapeutic interventions for liver diseases. The safety of PHF further supports its application, making it a promising candidate for effectively treating various liver conditions over different time scales.

[0421] More specifically, the above results indicate that the PHF according to the present application can effectively treat NAFLD, which is a disease continuum characterized by excessive lipid accumulation in hepatocytes. It progresses from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH), and in more severe cases, to liver fibrosis and cirrhosis.

[0422] The PHF according to the present application can also treat fatty liver disease, which is the most common chronic liver disease in Western societies. Fatty liver disease is not caused by alcohol or hepatitis viruses, but by the accumulation of fatty acids in the form of triglycerides in hepatocytes. In particular, histological fat accumulation in the liver is a characteristic of non-alcoholic fatty liver disease and presents various symptoms, including simple steatosis, steatohepatitis, and cirrhosis. In addition, fatty liver disease increases insulin resistance, thereby promoting the breakdown of adipose tissue. The resulting increased fatty acid supply to the liver promotes fatty acid oxidation, thereby inducing fat accumulation in hepatocytes.

[0423] The PHF according to the present application also provides protection against these processes. The content of MDA produced by lipid peroxidation in the liver was evaluated, and lipid peroxidation can directly damage hepatocytes, exacerbate liver inflammation, and mediate fibrosis.

[0424] The PHF according to the present application is also helpful for treating hepatic steatosis, which is a hallmark feature of NAFLD, whereby lipid droplets accumulate in hepatocytes in the form of triglycerides. When the accumulation occurs in >5% of hepatocytes, it is histologically diagnosed as NAFLD.

[0425] In addition to the above, the PHF according to the present application is active against the following conditions. The triglycerides in the liver with NAFLD are derived from the esterification of glycerol and free fatty acids (FFAs). Triglyceride accumulation occurs when the rate of FFA input or synthesis by hepatocytes exceeds the rate of output or catabolism. Obesity, especially insulin resistance (IR), is closely associated with the development of NAFLD. Overexpression of tumor necrosis factor (TNF)-α activates IκB kinase β, which plays an important role in the development of IR by inhibiting the phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2. IR leads to elevated hepatic triglyceride levels and ultimately hepatic steatosis through various mechanisms. Insulin fails to inhibit lipolysis in adipose tissue through hormone-sensitive lipase, resulting in an increase in FFAs flowing into the bloodstream, which are then taken up by the liver.

[0426] The PHF according to the present application is beneficial for the metabolic alterations of lipid profiles and glucose homeostasis and the functional and structural changes of hepatocytes in NAFLD induced by a high-fat diet. Feeding control animals with a high-fat diet results in a sharp increase in total cholesterol, triglycerides, and LDL-C, but a decrease in HDL-C levels. This hyperlipidemia may be related to enhanced de-esterification of abundant FFAs and reduced lipoproteins. In addition, the control animals showed enhanced oxidative stress, manifested as an increase in MDA levels, which is related to the β-oxidation of fatty acids in hepatic steatosis, and β-oxidation stimulates the production of reactive oxygen species, lipid peroxidation, hepatocyte necrotic inflammation, and apoptosis. The results indicate that administration of different doses of PHF can effectively alleviate NAFLD and abnormal lipid metabolism in high-fat diet-fed rats.

[0427] In vivo and in vitro studies on the toxicity of PHF were conducted. Test animals were fed with PHF for 60 days. All 17 organs of the animals were extracted and acute and subacute toxicity studies were performed. PHF is non-toxic to animals. The results show that PHF is safe when fed at a concentration of 2000 mg / kg / day.

[0428] Both DPPH and ABTS antioxidant analyses indicate that PHF capsules have antioxidant properties. The significant antioxidant activity of PHF capsules can potentially alleviate liver inflammation. In addition, PHF capsules have the potential to stimulate the secretion of IL2, IL4, and IFNγ cytokines in peripheral blood mononuclear cells (PBMCs).

[0429] The anti-HBV analysis of PHF showed that the PHF of the present invention has the potential to inhibit hepatitis B virus in vitro. The potential of PHF in treating liver fibrosis, liver cirrhosis, and non-alcoholic fatty liver was studied. Compared with untreated animals, the biochemical parameters of liver function tests in animals treated with PHF returned to normal. This indicates that PHF is effective in treating liver diseases such as liver fibrosis, liver cirrhosis, and non-alcoholic fatty liver. The same results were also observed in the tissues of the treatment group. Histopathological studies of the livers of treated animals showed that the liver structure was almost normal, without centrilobular necrosis and hepatic steatosis.

[0430] The Bliss independence model confirmed that the combination of RA, PA, and EA in PHF is more effective than individual components, making it a promising synergistic approach for treating liver fibrosis.

[0431] Therefore, this application shows that PHF powder has significant multi-factor effectiveness for hepatoprotective activity. This study claims the new use of PHF powder, that is, PHF powder has effective antioxidant properties, immunomodulatory properties, antiviral properties, anti-HBV properties, and can be used to treat liver fibrosis, liver cirrhosis, and / or NAFLD.

[0432] Specifically, this application (i.e., PHF) has the following effects:

[0433] The activity against NAFLD ranges from hepatic steatosis to NASH. The biochemical parameters of liver function return to normal levels. In addition, the livers of the subjects return to normal structure, with essentially no observable centrilobular necrosis and hepatic steatosis.

[0434] The activity against liver diseases includes hepatotoxicity, especially hepatotoxicity caused by one or more selected from the group consisting of alcohol, environmental pollution, virus-induced hepatitis, hepatocellular carcinoma, and hepatotoxic drugs. In addition, the PHF powder according to this application is active against virus-induced hepatitis caused by hepatitis B virus. In particular, if the oxidative damage is caused by oxidative stress or lipid peroxidation, especially by reactive oxygen species, the PHF powder is very active. Typical reactive oxygen species include one or more selected from the group consisting of hydrogen peroxide, superoxide anion radical, or hydroxyl radical.

[0435] The PHF powder of this application also provides hepatoprotective activity and antioxidant properties. The hepatoprotective activity can be demonstrated by a decrease in the level of one or more selected from the group consisting of AST, ALT, total bilirubin, and ALP. In addition, the hepatoprotective activity is manifested as an increase in the level of one or more selected from the group consisting of albumin, total protein, and globulin. In addition, the hepatoprotective activity is the result of the multi-herbal preparation regulating the integrity of the liver cell membrane due to its antioxidant properties.

[0436] In addition to the above, the PHF according to the present application reduces insulin resistance in subjects in need thereof. The PHF powder of the present application also shows that it does not significantly change the intake requirements of the subjects. In addition, it has been shown that, except for lymphocytes, the treatment with PHF involves no significant change in blood parameters. The PHF of the present application also has antioxidant properties and can be used in subjects. Typical antioxidant properties can reduce inflammation in the liver of the subjects. In addition, it has been demonstrated that the said PHF exhibits immunomodulatory properties and can stimulate the secretion of at least one of interleukin 2, interleukin 4 and interferon γ cytokines in the PBMCs of the subjects.

[0437] Finally, it has been shown that PHF has antiviral properties, especially anti-HBV properties, and it also shows activity against liver fibrosis, cirrhosis and NAFLD. In addition, when the said polyherbal preparation is administered to subjects, PHF increases secondary metabolites. Secondary metabolites are responsible for the antioxidant and anti-inflammatory properties of the subjects. In addition, PHF can be used to inhibit hepatitis B virus in subjects. Finally, PHF can be used to relieve NAFLD, abnormal lipid metabolism and / or excessive accumulation of lipids in hepatocytes in subjects in need thereof. In addition, PHF can reduce lipid accumulation in hepatocytes in the liver of the subjects, especially when the lipids contain triglycerides.

[0438] Although the above in-vivo experiments were conducted on mice, the volume of PHF can be adjusted according to the following formula for administration to humans:

[0439] <Human equivalent dose (mg / kg) = dose to be converted / (human Km / mouse Km)>.

[0440] Here, the Km factor is constant for a species with a specific body weight - that is, Km = body weight / body surface area.

[0441] Taking advantage of this, experiments on humans for the treatment of hepatitis B have been carried out, and the results are similar to those of the in-vivo experiments on mice.

[0442] These results indicate that the PHF powder has a wide range of applications and exhibits properties such as immunomodulatory properties, antiviral properties, and activities against non-alcoholic fatty liver disease, cirrhosis and fibrosis.

[0443] General disclosure in further exemplary embodiments:

[0444] Example 1: Use of a polyherbal preparation in the preparation of a pharmaceutical composition for treating liver diseases, liver injury or liver toxicity in subjects in need thereof, the polyherbal preparation comprising plant materials from two or more selected from the group consisting of Rauwolfia spp., Eclipta spp. and Phyllanthus spp.

[0445] Example 2: Use according to Example 1, wherein the Rauwolfia spp. includes Rauvolfia serpentina.

[0446] Example 3: Use according to any one of the foregoing examples, wherein the Eclipta spp. includes Eclipta prostrata.

[0447] Example 4: Use according to any one of the foregoing examples, wherein the Phyllanthus spp. includes Phyllanthus amarus.

[0448] Example 5: Use according to any one of the foregoing examples, wherein the plant material in the multi-herbal preparation comprises Rauvolfia serpentina, Eclipta prostrata, and Phyllanthus amarus.

[0449] Example 6: Use according to any one of the foregoing examples, wherein the multi-herbal preparation comprises about 40 - 50% of Eclipta prostrata.

[0450] Example 7: Use according to any one of the foregoing examples, wherein the multi-herbal preparation comprises about 20 - 30% of Phyllanthus amarus.

[0451] Example 8: Use according to any one of the foregoing examples, wherein the multi-herbal preparation comprises about 25 - 35% of Rauvolfia serpentina.

[0452] Example 9: Use according to any one of the foregoing examples, wherein the multi-herbal preparation comprises about 44 - 45% of Eclipta prostrata, 24 - 25% of Phyllanthus amarus, and 30 - 31% of Rauvolfia serpentina.

[0453] Example 10: Use according to any one of the foregoing examples, wherein a 100 mg preparation of the multi-herbal preparation comprises 44.75 mg of Eclipta prostrata, 24.58 mg of Phyllanthus amarus, and 30.66 mg of Rauvolfia serpentina.

[0454] Example 11: Use according to any one of the foregoing examples, wherein the pH of the multi-herbal preparation is about 4.5 to 6.5, more preferably 4.5 to 5.

[0455] Example 12: Use according to any one of the foregoing examples, wherein the multi-herbal preparation is dissolved in one or more selected from the group consisting of water, chloroform, ethanol, and ether.

[0456] Example 13: Use according to any one of the foregoing examples, wherein the content of phytochemical components in the multi-herbal preparation contains more than 35 mg / g of alkaloids, more preferably more than 45 mg / g of alkaloids.

[0457] Example 14: Use according to any one of the foregoing examples, wherein the phytochemical content of the polyherbal preparation comprises more than 110 mg / g flavonoids, more preferably more than 120 mg / g flavonoids.

[0458] Example 15: Use according to any one of the foregoing examples, wherein the phytochemical content of the polyherbal preparation comprises more than 40 mg / g total phenols, more preferably more than 50 mg / g total phenols.

[0459] Example 16: Use according to any one of the foregoing examples, wherein the phytochemical content of the polyherbal preparation comprises more than 20 mg / g tannins, more preferably more than 25 mg / g tannins.

[0460] Example 17: Use according to any one of the foregoing examples, wherein the phytochemical content of the polyherbal preparation comprises more than 60 mg / g saponins, more preferably more than 70 mg / g saponins.

[0461] Example 18: Use according to any one of the foregoing examples, wherein the phytochemical content of the polyherbal preparation comprises more than 10 mg / g total proteins, more preferably more than 15 mg / g total proteins.

[0462] Example 19: Use according to any one of the foregoing examples, wherein the polyherbal preparation comprises secondary metabolites, and the secondary metabolites comprise at least one of flavonoids, saponins, comestan, phenols, alkaloids, tannins and / or lignans.

[0463] Example 20: Use according to Example 19, wherein the most abundant secondary metabolites in the polyherbal preparation comprise flavonoids.

[0464] Example 21: Use according to any one of Examples 19 and 20, wherein the flavonoids comprise one or more selected from the group consisting of luteolin-7-glucoside, luteolin, apigenin and Orobol (isoluteolin).

[0465] Example 22: Use according to any one of Examples 19 and 21, wherein the flavonoids providing hepatoprotective effects comprise one or more selected from the group consisting of luteolin and apigenin.

[0466] Example 23: Use according to any one of Examples 19 to 22, wherein the flavonoids providing antioxidant activity comprise one or more selected from the group consisting of luteolin-7-glucoside, luteolin, apigenin and Orobol (isoluteolin).

[0467] Example 24: Use according to Example 19, wherein the comitans providing a hepatoprotective effect comprises one or more selected from the group consisting of wedelolactone, dehydrowedelolactone, dehydrowedelolactone-7-glucoside.

[0468] Example 25: Use according to Example 19, wherein the tannins comprise one or more selected from the group consisting of geraniin, corilagin, geraniinic acid, amarinic acid and elaeocarpusin.

[0469] Example 26: Use according to any one of Examples 19 and 25, wherein the tannins providing a hepatoprotective effect comprise one or more selected from the group consisting of corilagin, gallic acid and amariin.

[0470] Example 27: Use according to any one of Examples 19, 25 and 26, wherein the tannins providing antioxidant activity comprise one or more selected from the group consisting of corilagin and geraniin.

[0471] Example 28: Use according to Example 19, wherein the lignans comprise one or more selected from the group consisting of phyllanthin, hypophyllanthin, lintetralin, niranthin, nirtetralin, isolintetralin and isonirtetralin.

[0472] Example 29: Use according to any one of Examples 19 and 28, wherein the lignans providing a hepatoprotective effect comprise one or more selected from the group consisting of phyllanthin and hypophyllanthin.

[0473] Example 30: Use according to any one of Examples 19, 28 and 29, wherein the lignans providing antioxidant activity comprise one or more selected from the group consisting of phyllanthin and hypophyllanthin.

[0474] Example 31: Use according to Example 19, wherein the alkaloids comprise one or more selected from the group consisting of reserpine, rescinnamine and serpentine.

[0475] Example 32: Use according to any one of the foregoing examples, wherein the multi-herbal preparation is sterilized by microwave pasteurization and / or ultraviolet radiation.

[0476] Example 33: Use according to any one of the preceding examples, wherein the liver disease comprises NAFLD, cirrhosis, fibrosis or anti-HBV.

[0477] Example 34: Use according to Example 33, wherein the NAFLD comprises the range from hepatic steatosis to NASH.

[0478] Example 35: Use according to any one of the preceding examples, wherein the biochemical parameters of liver function are restored to normal levels.

[0479] Example 36: Use according to any one of the preceding examples, wherein the liver of the subject is restored to a normal structure, with substantially no observable centrilobular necrosis and hepatic steatosis.

[0480] Example 37: Use according to any one of the preceding examples, wherein the liver disease is caused by hepatotoxicity.

[0481] Example 38: Use according to any one of the preceding examples, wherein the hepatotoxicity is caused by one or more selected from the group consisting of alcohol, environmental pollution, virus-induced hepatitis, hepatocellular carcinoma and hepatotoxic drugs.

[0482] Example 39: Use according to Example 38, wherein the virus-induced hepatitis is caused by hepatitis A virus, hepatitis B virus or hepatitis C virus.

[0483] Example 40: Use according to any one of the preceding examples, wherein the liver injury is caused by oxidative damage or liver inflammation.

[0484] Example 41: Use according to Example 40, wherein the oxidative damage is caused by oxidative stress or lipid peroxidation.

[0485] Example 42: Use according to Example 41, wherein the oxidative stress and lipid peroxidation are caused by reactive oxygen species.

[0486] Example 43: Use according to Example 42, wherein the reactive oxygen species comprise one or more substances selected from the group consisting of hydrogen peroxide, superoxide anion radical and hydroxyl radical.

[0487] Example 44: A multi-herbal preparation comprising or consisting of the following ingredients:

[0488] a) a plant or plant part of the Eclipta family, in particular a plant or plant part of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0489] b) Plants or plant parts of the Phyllanthaceae family, especially plants or plant parts of Phyllanthus amarus, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0490] c) Plants or plant parts of the Apocynaceae family, especially plants or plant parts of Rauvolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0491] Example 45: The multi-herbal preparation according to Example 44, which comprises not more than 70%, preferably not more than 60%, of plants or plant parts of the Ecliptaceae family.

[0492] Example 46: The multi-herbal preparation according to Example 44 or 45, which contains not more than 30% of plants or plant parts of the Phyllanthaceae family.

[0493] Example 47: The multi-herbal preparation according to any one of Examples 44 to 46, which contains not more than 36% of plants or plant parts of the Apocynaceae family.

[0494] Example 48: The multi-herbal preparation according to any one of Examples 44 to 47, which contains 43% to 46% of plants or plant parts of the Ecliptaceae family, 23% to 26% of plants or plant parts of the Phyllanthaceae family, and 29% to 32% of plants or plant parts of the Apocynaceae family.

[0495] Example 49: The multi-herbal preparation according to any one of Examples 44 to 48, wherein 100 mg of PHF contains 44.5 mg to 44.85 mg of plants or plant parts of the Ecliptaceae family, 24.5 mg to 24.6 mg of plants or plant parts of the Phyllanthaceae family, and 30.6 mg to 30.7 mg of plants or plant parts of the Apocynaceae family.

[0496] Example 50: The multi-herbal preparation according to any one of Examples 44 to 49, wherein the plants or plant parts are plants or plant parts of Eclipta prostrata, Phyllanthus amarus, and Rauvolfia serpentina.

[0497] Example 51: The multi-herbal preparation according to any one of Examples 44 to 50, wherein the plant parts are selected from one or more of fruits, seeds, rhizomes, buds, leaves, husks, barks, flower petals and flower petal extracts, peels, seed coats, and stems.

[0498] Example 52: The multi-herbal preparation according to any one of Examples 44 to 51, which further comprises one or more secondary metabolites selected from flavonoids, saponins, comestans, phenols, alkaloids, tannins, and lignans.

[0499] Example 53: A multi-herbal preparation according to any one of Examples 44 to 52, wherein the phytochemical content of the multi-herbal preparation comprises more than 35 mg / g of alkaloids, more than 110 mg / g of flavonoids, more than 40 mg / g of total phenols, more than 20 mg / g of tannins, more than 60 mg / g of saponins, and more than 10 mg / g of total proteins.

[0500] Example 54: A liver-specific multi-herbal preparation comprising or consisting of the following ingredients:

[0501] a) A plant or plant part of the Ecliptaceae family, in particular a plant or plant part of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0502] b) A plant or plant part of the Phyllanthaceae family, in particular a plant or plant part of Phyllanthus niruri, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0503] c) A plant or plant part of the Apocynaceae family, in particular a plant or plant part of Rauvolfia serpentina, in an amount of at least 25%, preferably at least 27%.

[0504] Example 55: A liver multi-herbal preparation comprising or consisting of the following ingredients:

[0505] a) A plant or plant part of the Ecliptaceae family, in particular a plant or plant part of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and

[0506] b) A plant or plant part of the Phyllanthaceae family, in particular a plant or plant part of Phyllanthus niruri, in an amount of at least 15%, preferably at least 20% and at most 40%; and

[0507] c) A plant or plant part of the Apocynaceae family, in particular a plant or plant part of Rauvolfia serpentina, in an amount of at least 25%, preferably at least 27%.

Claims

1. A multi-herb preparation comprising or consisting of the following ingredients: a) plants or plant parts of the family Channataceae, in particular plants or plant parts of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and b) plants or plant parts of the family Phyllanthaceae, in particular Phyllanthus urinaria, in an amount of at least 15%, preferably at least 20% and at most 40%; and c) Plants or plant parts of the family Rauvolfaceae, in particular plants or plant parts of Rauwolfia serpentina, in an amount of at least 25%, preferably at least 27%.

2. A multi-herb preparation according to claim 1 comprising not more than 70%, and preferably not more than 60%, of plants or plant parts of the Channatiaceae family.

3. A multi-herb preparation according to claim 1 or 2 comprising not more than 30% of plants or plant parts of the family Phyllanthaceae.

4. A multi-herb preparation according to any one of claims 1 to 3 comprising not more than 36% of plants or plant parts of the family Rauvolfaceae.

5. A multi-herb preparation according to any one of claims 1 to 4, comprising 43% to 46% of plants or plant parts of the Channatiaceae family, 23% to 26% of plants or plant parts of the Phyllanthaceae family and 29% to 32% of plants or plant parts of the Rauvolfaceae family.

6. The multi-herb preparation according to any one of claims 1 to 5, wherein 100 mg of the multi-herb preparation comprises 44.5 mg to 44.85 mg of plants or plant parts of the Channatiaceae family, 24.5 mg to 24.6 mg of plants or plant parts of the Phyllanthaceae family and 30.6 mg to 30.7 mg of plants or plant parts of the Rauvolfaceae family.

7. The multi-herb preparation according to any one of claims 1 to 6, wherein the plants or plant parts are plants or plant parts of Eclipta prostrata, Phyllanthus urticae and Rauwolfia serrulata.

8. The multi-herb preparation according to any one of claims 1 to 7, wherein the plant parts are selected from one or more of fruits, seeds, rhizomes, buds, leaves, husks, bark, petals and petal extracts, pericarps, seed coats and stems.

9. The multi-herb preparation according to any one of claims 1 to 8, further comprising one or more secondary metabolites selected from the group consisting of flavonoids, saponins, comistans, phenols, alkaloids, tannins and lignans.

10. The multi-herb preparation according to any one of claims 1 to 9, wherein the phytochemical content of the multi-herb preparation comprises greater than 35 mg / g alkaloids, greater than 110 mg / g flavonoids, greater than 40 mg / g total phenols, greater than 20 mg / g tannins, greater than 60 mg / g saponins and greater than 10 mg / g total protein.

11. Use of a multi-herb preparation according to any one of claims 1 to 10 for the preparation of a medicament for treating liver disease, liver damage or liver toxicity in a subject in need thereof.

12. The use according to claim 11, wherein the liver disease comprises non-alcoholic fatty liver disease (NAFLD), cirrhosis, fibrosis or HBV infection.

13. The use according to claim 12, wherein the liver disease is caused by hepatotoxicity, and wherein the hepatotoxicity is caused by one or more selected from the group consisting of alcohol, environmental pollution, virus-induced hepatitis, hepatocellular carcinoma and hepatotoxic drugs.

14. The use according to claim 13, wherein the virus-induced hepatitis is caused by hepatitis A virus, hepatitis B virus or hepatitis C virus.

15. A liver-specific multi-herb preparation comprising or consisting of: a) plants or plant parts of the family Channataceae, in particular plants or plant parts of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and b) plants or plant parts of the family Phyllanthaceae, in particular plants or plant parts of bitter Phyllanthus urinaria, in an amount of at least 15%, preferably at least 20% and at most 40%; and c) Plants or plant parts of the family Rauvolfaceae, in particular plants or plant parts of Rauwolfia serpentina, in an amount of at least 25%, preferably at least 27%.

16. A liver multi-herb preparation comprising or consisting of the following ingredients: a) plants or plant parts of the family Channataceae, in particular plants or plant parts of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and b) plants or plant parts of the family Phyllanthaceae, in particular plants or plant parts of bitter Phyllanthus urinaria, in an amount of at least 15%, preferably at least 20% and at most 40%; and c) Plants or plant parts of the family Rauvolfaceae, in particular plants or plant parts of Rauwolfia serpentina, in an amount of at least 25%, preferably at least 27%.

17. Use of a multi-herbal preparation for the preparation of a medicament for treating hepatitis B virus (HBV) infection in a subject in need thereof, wherein the multi-herbal preparation of the medicament comprises or consists of the following ingredients: a) plants or plant parts of the family Channataceae, in particular plants or plant parts of Eclipta prostrata, in an amount of at least 30%, preferably at least 35% or 40%; and b) plants or plant parts of the family Phyllanthaceae, in particular plants or plant parts of bitter Phyllanthus urinaria, in an amount of at least 15%, preferably at least 20% and at most 40%; and c) Plants or plant parts of the family Rauvolfaceae, in particular plants or plant parts of Rauwolfia serpentina, in an amount of at least 25%, preferably at least 27%.

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